Microarray Data Analysis

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Microarray Data Analysis

Class discovery and Class prediction: Clustering and Discrimination

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Gene expression profiles

• Many genes show definite changes of expression between conditions

• These patterns are called gene profiles

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Motivation (1): The problem of finding patterns

• It is common to have hybridizations where conditions reflect temporal or spatial aspects.– Yeast cycle data– Tumor data evolution after chemotherapy– CNS data in different part of brain

• Interesting genes may be those showing patterns associated with changes.

• Our problem seems to be distinguishing interesting or real patterns from meaningless variation, at the level of the gene

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Finding patterns: Two approaches

• If patterns already exist Profile comparison (Distance analysis)– Find the genes whose expression fits specific,

predefined patterns.– Find the genes whose expression follows the

pattern of predefined gene or set of genes.

• If we wish to discover new patterns Cluster analysis (class discovery)– Carry out some kind of exploratory analysis to

see what expression patterns emerge;

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Motivation (2): Tumor classification

• A reliable and precise classification of tumours is essential for successful diagnosis and treatment of cancer.

• Current methods for classifying human malignancies rely on a variety of morphological, clinical, and molecular variables.

• In spite of recent progress, there are still uncertainties in diagnosis. Also, it is likely that the existing classes are heterogeneous.

• DNA microarrays may be used to characterize the molecular variations among tumours by monitoring gene expression on a genomic scale. This may lead to a more reliable classification of tumours.

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Tumor classification, cont

• There are three main types of statistical problems associated with tumor classification:

1. The identification of new/unknown tumor classes using gene expression profiles - cluster analysis;

2. The classification of malignancies into known classes - discriminant analysis;

3. The identification of “marker” genes that characterize the different tumor classes - variable selection.

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Cluster and Discriminant analysis

• These techniques group, or equivalently classify, observational units on the basis of measurements.

• They differ according to their aims, which in turn depend on the availability of a pre-existing basis for the grouping.– In cluster analysis (unsupervised learning, class

discovery) , there are no predefined groups or labels for the observations,

– Discriminant analysis (supervised learning, class prediction) is based on the existence of groups (labels)

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Clustering microarray data

• Cluster can be applied to genes (rows), mRNA samples (cols), or both at once.– Cluster samples to identify new cell or tumour

subtypes.– Cluster rows (genes) to identify groups of co-

regulated genes.– We can also cluster genes to reduce

redundancy e.g. for variable selection in predictive models.

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Advantages of clustering

• Clustering leads to readily interpretable figures.• Clustering strengthens the signal when averages

are taken within clusters of genes (Eisen).• Clustering can be helpful for identifying patterns

in time or space. • Clustering is useful, perhaps essential, when

seeking new subclasses of cell samples (tumors, etc).

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Applications of clustering (1)

– Alizadeh et al (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

• Three subtypes of lymphoma (FL, CLL and DLBCL) have different genetic signatures. (81 cases total)

• DLBCL group can be partitioned into two subgroups with significantly different survival. (39 DLBCL cases)

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Clusters on both genes and arrays

Taken from Nature February, 2000Paper by Allizadeh. A et alDistinct types of diffuse large B-cell lymphoma identified by Gene expression profiling,

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Discovering tumor subclasses

• DLBCL is clinically heterogeneous

• Specimens were clustered based on their expression profiles of GC B-cell associated genes.

• Two subgroups were discovered:– GC B-like DLBCL– Activated B-like

DLBCL

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Applications of clustering (2)

• A naïve but nevertheless important application is assessment of experimental design

• If one has an experiment with different experimental conditions, and in each of them there are biological and technical replicates…

• We would expect that the more homogeneous groups tend to cluster together– Tech. replicates < Biol. Replicates < Different groups

• Failure to cluster so suggests bias due to experimental conditions more than to existing differences.

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Basic principles of clustering

Aim: to group observations that are “similar” based on predefined criteria.

Issues: Which genes / arrays to use?

Which similarity or dissimilarity measure?

Which clustering algorithm?

• It is advisable to reduce the number of genes from the full set to some more manageable number, before clustering. The basis for this reduction is usually quite context specific, see later example.

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Two main classes of measures of dissimilarity

• Correlation

• Distance

– Manhattan

– Euclidean

– Mahalanobis distance

– Many more ….

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Two basic types of methods

Partitioning Hierarchical

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Partitioning methods

Partition the data into a pre-specified number k of mutually exclusive and exhaustive groups.

Iteratively reallocate the observations to clusters until some criterion is met, e.g. minimize within cluster sums of squares.

Examples:– k-means, self-organizing maps (SOM), PAM, etc.;– Fuzzy: needs stochastic model, e.g. Gaussian

mixtures.

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Hierarchical methods

• Hierarchical clustering methods produce a tree or dendrogram.

• They avoid specifying how many clusters are appropriate by providing a partition for each k obtained from cutting the tree at some level.

• The tree can be built in two distinct ways– bottom-up: agglomerative clustering;– top-down: divisive clustering.

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Agglomerative methods

• Start with n clusters.• At each step, merge the two closest clusters

using a measure of between-cluster dissimilarity, which reflects the shape of the clusters.

• Between-cluster dissimilarity measures– Mean-link: average of pairwise dissimilarities– Single-link: minimum of pairwise

dissimilarities.– Complete-link: maximum& of pairwise

dissimilarities.– Distance between centroids

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Distance between centroids Single-link

Complete-link Mean-link

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Divisive methods

• Start with only one cluster.• At each step, split clusters into two parts.• Split to give greatest distance between two new

clusters• Advantages.

• Obtain the main structure of the data, i.e. focus on upper levels of dendogram.

• Disadvantages. – Computational difficulties when considering all

possible divisions into two groups.

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1 5 2 3 4

1 5 2 3 4

1,2,5

3,41,5

1,2,3,4,5

Agglomerative

Illustration of points In two dimensional space

1

5

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2

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1 5 2 3 4

1 5 2 3 4

1,2,5

3,41,5

1,2,3,4,5

Agglomerative

Tree re-ordering?

1

5

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2

1 52 3 4

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Partitioning or Hierarchical?

• Partitioning:– Advantages

• Optimal for certain criteria.• Genes automatically

assigned to clusters

– Disadvantages• Need initial k;• Often require long

computation times.• All genes are forced into a

cluster.

• Hierarchical– Advantages

• Faster computation.• Visual.

– Disadvantages• Unrelated genes are

eventually joined• Rigid, cannot correct

later for erroneous decisions made earlier.

• Hard to define clusters.

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Hybrid Methods

• Mix elements of Partitioning and Hierarchical methods– Bagging

• Dudoit & Fridlyand (2002)

– HOPACH • van der Laan & Pollard (2001)

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Three generic clustering problems

Three important tasks (which are generic) are:

1. Estimating the number of clusters;2. Assigning each observation to a cluster;3. Assessing the strength/confidence of

cluster assignments for individual observations.

Not equally important in every problem.

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Estimating number of clusters using silhouette

• Define silhouette width of the observation as :

S = (b-a)/max(a,b) • Where a is the average dissimilarity to all the points in the cluster

and b is the minimum distance to any of the objects in the other clusters.

• Intuitively, objects with large S are well-clustered while the ones with small S tend to lie between clusters.

• How many clusters: Perform clustering for a sequence of the number of clusters k and choose the number of components corresponding to the largest average silhouette.

• Issue of the number of clusters in the data is most relevant for novel class discovery, i.e. for clustering samples

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Estimating number of clusters using the bootstrap

There are other resampling (e.g. Dudoit and Fridlyand, 2002) and non-resampling based rules for estimating the number of clusters (for review see Milligan and Cooper (1978) and Dudoit and Fridlyand (2002) ).

The bottom line is that none work very well in complicated situation and, to a large extent, clustering lies outside a usual statistical framework.

It is always reassuring when you are able to characterize a newly discovered clusters using information that was not used for clustering.

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LimitationsCluster analyses:• Usually outside the normal framework of statistical

inference;• less appropriate when only a few genes are likely to

change.• Needs lots of experiments• Always possible to cluster even if there is nothing going

on.• Useful for learning about the data, but does not provide

biological truth.

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Discrimination

or Class prediction

or Supervised Learning

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Motivation: A study of gene expression on breast tumours

(NHGRI, J. Trent)• How similar are the gene

expression profiles of BRCA1 and BRCA2 (+) and sporadic breast cancer patient biopsies?

• Can we identify a set of genes that distinguish the different tumor types?

• Tumors studied:– 7 BRCA1 +– 8 BRCA2 +– 7 Sporadic

cDNA MicroarraysParallel Gene Expression Analysis

6526 genes /tumor

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Discrimination• A predictor or classifier for K tumor classes partitions the space X of

gene expression profiles into K disjoint subsets, A1, ..., AK, such that for a sample with expression profile x=(x1, ...,xp) Ak the predicted class is k.

• Predictors are built from past experience, i.e., from observations which are known to belong to certain classes. Such observations comprise the learning set

L = (x1, y1), ..., (xn,yn).

• A classifier built from a learning set L is denoted by C( . ,L): X {1,2, ... ,K},

with the predicted class for observation x being C(x,L).

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Discrimination and Allocation

Learning SetData with

known classes

ClassificationTechnique

Classificationrule

Data with unknown classes

ClassAssignment

Discrimination

Prediction

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?Bad prognosis

recurrence < 5yrsGood Prognosis

recurrence > 5yrs

ReferenceL van’t Veer et al (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature, Jan..

ObjectsArray

Feature vectorsGene

expression

Predefine classesClinical

outcome

new array

Learning set

Classificationrule

Good PrognosisMatesis > 5

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B-ALL T-ALL AML

ReferenceGolub et al (1999) Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286(5439): 531-537.

ObjectsArray

Feature vectorsGene

expression

Predefine classes

Tumor type

?

new array

Learning set

ClassificationRule

T-ALL

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Components of class prediction

• Choose a method of class prediction– LDA, KNN, CART, ....

• Select genes on which the prediction will be base: Feature selection– Which genes will be included in the model?

• Validate the model– Use data that have not been used to fit the

predictor

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Prediction methods

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Choose prediction model

• Prediction methods – Fisher linear discriminant analysis (FLDA) and

its variants• (DLDA, Golub’s gene voting, Compound covariate

predictor…)

– Nearest Neighbor– Classification Trees– Support vector machines (SVMs)– Neural networks– And many more …

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Fisher linear discriminant analysis First applied in 1935 by M. Barnard at the suggestion of R. A.

Fisher (1936), Fisher linear discriminant analysis (FLDA) consists of

i. finding linear combinations x a of the gene expression profiles x=(x1,...,xp) with large ratios of between-groups to within-groups sums of squares - discriminant variables;

ii. predicting the class of an observation x by the class whose mean vector is closest to x in terms of the discriminant variables.

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FLDA

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Classification with SVMsGeneralization of the ideas of separating hyperplanes in the original space.Linear boundaries between classes in higher-dimensional space lead tothe non-linear boundaries in the original space.

Adapted from internet

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Nearest neighbor classification

• Based on a measure of distance between observations (e.g. Euclidean distance or one minus correlation).

• k-nearest neighbor rule (Fix and Hodges (1951)) classifies an observation x as follows:– find the k observations in the learning set closest to x– predict the class of x by majority vote, i.e., choose

the class that is most common among those k observations.

• The number of neighbors k can be chosen by cross-validation (more on this later).

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Nearest neighbor rule

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Classification tree

• Binary tree structured classifiers are constructed by repeated splits of subsets (nodes) of the measurement space X into two descendant subsets, starting with X itself.

• Each terminal subset is assigned a class label and the resulting partition of X corresponds to the classifier.

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Classification treesMi1 < 1.4Node 1Class 1: 10Class 2: 10

Mi2 > -0.5Node 2Class 1: 6Class 2: 9

Node 4Class 1: 0Class 2: 4Prediction: 2


yes

yes

no

noGene 1

Gene 2

Mi2 > 2.1Node 5Class 1: 6Class 2: 5



Gene 3

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Three aspects of tree construction

• Split selection rule: – Example, at each node, choose split maximizing decrease in

impurity (e.g. Gini index, entropy, misclassification error).

• Split-stopping: The decision to declare a node as terminal or to continue splitting.

– Example, grow large tree, prune to obtain a sequence of subtrees, then use cross-validation to identify the subtree with lowest misclassification rate.

• The assignment: of each terminal node to a class

– Example, for each terminal node, choose the class minimizing the resubstitution estimate of misclassification probability, given that a case falls into this node.

Supplementary slide

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Other classifiers include…

• Support vector machines

• Neural networks

• Bayesian regression methods

• Projection pursuit

• ....

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Aggregating predictors

• Breiman (1996, 1998) found that gains in accuracy could be obtained by aggregating predictors built from perturbed versions of the learning set.

• In classification, the multiple versions of the predictor are aggregated by voting.

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Another component in classification rules:aggregating classifiers

Training Set

X1, X2, … X100

Classifier 1Resample 1




Examples:BaggingBoosting

Random Forest

Aggregateclassifier

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Aggregating classifiers:Bagging

Training Set (arrays)X1, X2, … X100

Tree 1Resample 1

X*1, X*2, … X*100

Lets the treevote

Tree 2Resample 2

X*1, X*2, … X*100

Tree 499Resample 499X*1, X*2, … X*100

Tree 500Resample 500X*1, X*2, … X*100

Testsample

Class 1

Class 2

Class 1

Class 1

90% Class 110% Class 2

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Feature selection

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Feature selection

• A classification rule must be based on a set of variables which contribute useful information for distinguishing the classes.

• This set will usually be small because most variables are likely to be uninformative.

• Some classifiers (like CART) perform automatic feature selection whereas others, like LDA or KNN, do not.

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Approaches to feature selection

• Filter methods perform explicit feature selection prior to building the classifier.– One gene at a time: select features based on the

value of an univariate test.– The number of genes or the test p-value are the

parameters of the FS method.• Wrapper methods perform FS implicitly, as a

part of the classifier building.– In classification trees features are selected at each

step based on reduction in impurity.– The number of features is determined by pruning the

tree using cross-validation.

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Why select features

• Lead to better classification performance by removing variables that are noise with respect to the outcome

• May provide useful insights into etiology of a disease.

• Can eventually lead to the diagnostic tests (e.g., “breast cancer chip”).

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Why select features?

Correlation plotData: Leukemia, 3 class

No feature selection

Top 100 feature selection

Selection based on variance

-1 +1

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Performance assessment

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• Before using a classifier for prediction or prognostic one needs a measure of its accuracy.

• The accuracy of a predictor is usually measured by the Missclassification rate: The % of individuals belonging to a class which are erroneously assigned to another class by the predictor.

• An important problem arises here– We are not interested in the ability of the predictor for classifying

current samples– One needs to estimate future performance based on what is

available.

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Estimating the error rate

• Using the same dataset on which we have built the predictor to estimate the missclassification rate may lead to erroneously low values due to overfitting.– This is known as the resubstitution estimator

• We should use a completely independent dataset to evaluate the classifier, but it is rarely available.

• We use alternatives approaches such as– Test set estimator– Cross validation

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Performance assessment (I)

• Resubstitution estimation: Compute the error rate on the learning set.– Problem: downward bias

• Test set estimation: Proceeds in two steps

1. Divide learning set into two sub-sets, L and T;

2. Build the classifier on L and compute error rate on T.

– This approach is not free from problems

• L and T must be independent and identically distributed.

• Problem: reduced effective sample size

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Diagram of performance assessment (I)

Resubstitution estimation

Training set


TrainingSet

Independenttest set

Classifier

Classifier

Test set estimation

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Performance assessment (II)

• V-fold cross-validation (CV) estimation: Cases in learning set randomly divided into V subsets of (nearly) equal size. Build classifiers by leaving one set out; compute test set error rates on the left out set and averaged. – Bias-variance tradeoff: smaller V can give larger bias but smaller

variance– Computationally intensive.

• Leave-one-out cross validation (LOOCV).

– Special case for V=n.

– Works well for stable classifiers (k-NN, LDA, SVM)

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Diagram of performance assessment (II)

Training set


TrainingSet

Independenttest set

(CV) Learningset

(CV) Test set

Classifier

Classifier

Classifier

Resubstitution estimation

Test set estimation

Cross Validation

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Examples

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Reference 1Retrospective studyL van’t Veer et al Gene expression profiling predicts clinical outcome of breast cancer. Nature, Jan 2002..

Learning set

Bad Good

ClassificationRule

Reference 2Cohort studyM Van de Vijver et al. A gene expression signature as a predictor of survival in breast cancer. The New England Jouranl of Medicine, Dec 2002.

Reference 3Prospective trials.Aug 2003Clinical trialshttp://www.agendia.com/

Feature selection.Correlation with class

labels, very similar to t-test.

Using cross validation toselect 70 genes

295 samples selected from Netherland Cancer Institute

tissue bank (1984 – 1995).

Results” Gene expression profile is a morepowerful predictor then standard systems based on clinical and histologic criteria

Agendia (formed by reseachers from the Netherlands Cancer Institute)Has started in Oct, 2003

1) 5000 subjects [Health Council of the Netherlands]2) 5000 subjects New York based Avon Foundation.

Custom arrays are made by Agilent including 70 genes + 1000 controls

Case studies

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Van’t Veer breast cancer study study

Investigate whether tumor ability for metastasis is

obtained later in development or inherent in the initial

gene expression signature.

• Retrospective sampling of node-negative women: 44 non-recurrences within 5 years of surgery and 34 recurrences. Additionally, 19 test sample (12 recur. and 7 non-recur)

• Want to demonstrate that gene expression profile is significantly associated with recurrence independent of the other clinical variables.

Nature, 2002

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Predictor development• Identify a set of genes with correlation > 0.3 with the binary outcome. Show that there

are significant enrichment for such genes in the dataset.• Rank-order genes on the basis of their correlation• Optimize number of genes in the classifier by using CV-1

Classification is made on the basis of the correlations of the expression profile of leave-out-out sample with the mean expression of the remaining samples from the good and bad prognosis patients, respectively.

N. B.: The correct way to select genes is within rather than outside cross-validation, resulting in different set of markers for each CV iteration

N. B. : Optimizing number of variables and other parameters should be done via 2-level cross-validation if results are to be assessed on the training set.

The classification indicator is included into the logistic model along with other clinical variables. It is shown that gene expression profile has the strongest effect. Note that some of this may be due to overfitting for the threshold parameter.

73Van ‘t Veer, et al., 2002

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van de Vuver’s breast data(NEJM, 2002)

• 295 additional breast cancer patients, mix of node-negative and node-positive samples.

• Want to use the predictor that was developed to identify patients at risk for metastasis.

• The predicted class was significantly associated with time to recurrence in the multivariate cox-proportional model.

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Acknowledgments

• Many of the slides in this course notes are based on web materials made available by their authors.

• I wish to thank specially– Yee Hwa Yang (UCSF), – Ben Boldstat, Sandrine Dudoit & Terry Speed, U.C.

Berkeley.– The Bioconductor Project– "Estadística I Bioinformàtica" research group at the

University of Barcelona

Microarray Data Analysis

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